The present invention relates primarily to a method of obtaining fibrinogen. The method of the present invention also enables the obtaining of fibronectin and Factor XIII.
The isolation of human fibrinogen has traditionally been carried out by classical plasma fractionation methods. Fibrinogen is precipitated from plasma either with ethanol (Blomback and Blomback, 1956), ammonium sulphate (Takeda, 1996), xcex2 alanine/glycine (Jakobsen and Kieruif, 1976), polymers (polyethelene glycol) and low ionic strength solutions (Holm, 1985) with relative high yield and homogeneity.
Further purification of fibrinogen precipitates can be achieved by ion-exchange chromatography conditions (Stathakis, 1978) and affinity chromatography (Kuyas, 1990). Specific contaminants can be absorbed out for example fibronectin an immobilised gelatine and plasminogen an immobilised lysine (Vuento, 1979).
Over the past few decades the overall structure and function of the fibrinogen molecule has been elucidated. The completion of the amino acid sequence of human fibrinogen (Henschen and Lottspeich, 1977) and the assignment of disulphide bonds (Blomback et al. 1976, Douma et al. 1978) provided data that confirmed the pioneer observations of an extended multidomained molecule (Hah and Slayter, 1959). The cloning of the fibrinogen genes and the complete amino acid sequence of all three chains of human fibrinogen from the cDNA studies are in agreement with those reported earlier based on the conventional amino acid sequencing procedures (Chung et al. 1983).
Precipitation methods are widely used for the manufacture of commercial fibrinogen. Chromatographic methods are now being explored as an alternative or to improve the purity of fibrinogen concentrates.
Fibrinogen interacts with a number of physiologically important proteins (Doolittle, 1984) such a plasminogen, thrombin, fibronectin, certain strains of staphylococcal bacteria and platelets. A number of functional characteristics have been assigned to specific parts of the molecule including: the position of the fibrinopeptides released from the parent molecule by catalytic action of thrombin, fibrin covalent stabilisation donor and acceptor sites, carbohydrate clusters, polymerisation sites, calcium binding sites and the attachment sites for fibronectin, plasminogen, bacteria and platelets.
Human fibrinogen has a strong affinity for fibrin and this association has been exploited to affinity purify fibrinogen. Fibrin immobilised on Sepharose was used to isolate fibrinogen from human plasma (Matthias et al. 1975). Protein structure function studies have identified the peptide sequences in fibrin that have been shown to specifically bind to fibrinogen. Short peptides beginning with the sequence Gly-L-Pro-Arg have been shown to bind fibrinogen (Laudano and Doolittle, 1978). This sequence corresponds to the first three amino acids of the fibrin xcex1-chain exposed by the thrombin catalysed release of the fibrinopeptide A in all vertebrate species. The addition of a second proline to this sequence was later shown to increase the affinity of the peptide Gly-Pro-Arg-Pro for fibrinogen almost ten-fold (Laudano and Doolittle, 1980). Based on this information, synthetic peptides corresponding to these sequences have also been shown to bind to fibrinogen (Gartner and Taylor, 1991).
The present invention relates to the large scale separation by precipitation of fibrinogen from other blood proteins in human blood plasma, cryoprecipitate, fraction 1 precipitate, other plasma fractions containing fibrinogen or fibrinogen containing culture media produced by recombinant DNA techniques and subsequent treatment of the heparin precipitate. The resultant fibrinogen-enriched preparation may be further purified to homogeneity utilising other precipitation methods, chromatographic steps such as ion-exchange chromatography, affinity chromatography size exclusion chromatography or ultrafiltration.
The present inventors have found that fibrinogen may be recovered from heparin precipitated paste, a by-product from the manufacturing process of Factor VIII (Antihaemophilic Factor, AHF). The heparin precipitate paste may be solubilised with salt containing solutions such as NaCl to provide a fibrinogen preparation of high specific activity. The method of this invention has been shown to be superior to other known isolation procedures in that fibrinogen may be obtained with relative high yield and homogeneity from a discard fraction of processed plasma.
Accordingly, the present invention consists in a method of obtaining a fibrinogen enriched preparation, the method including the following steps:
(i) adding an effective amount of a sulphated polysaccharide (SPS) to a fibrinogen containing solution to form a fibrinogen containing percipitate; and
(ii) extracting fibrinogen from the fibrinogen containing percipitate from step (i) with a solution containing at least 0.1 M, and preferably at least 0.2M, salt to obtain a fibrinogen enriched preparation.
In a preferred embodiment of the present invention the solution includes at least one salt selected from the group consisting of chloride, phosphate and acetate salts, and more preferably includes NaCl. It is preferred that the NaCl is present at concentration of from about 0.1M to about 2.0M, preferably from about 0.2M to about 0.8M.
In a further preferred embodiment the solution includes xcex5-aminocaproic acid.
In another preferred embodiment the SPS is a heparinoid selected from the group consisting of mucopolysaccharide polysulphate, pentosan polysulphate, chondroitin sulphate, dextran sulphate and heparin and is preferably heparin.
The amount of SPS used can be readily determined, however, it is preferred that the SPS is added to the fibrinogen containing solution to provide a concentration of SPS of at least 0.15 mg/ml.
Where the fibrinogen is to be used therapeutically the fibrinogen will be subjected to a viral inactivation step(s). Such inactivation procedures are well known in the art and include heating and solvent detergent treatment.
The fibrinogen containing solution may be any of a number of such solutions well known to those skilled in the art such as plasma (including anti-coagulated plasma), plasma fractions (such as cryoprecipitate and solubilised fraction I) and fibrinogen-containing cell culture media arising from the production of fibrinogen by recombinant DNA techniques. It is, however, preferred that the fibrinogen containing solution is a blood plasma fraction, preferably cryoprecipitate.
The fibrinogen may be further purified from the fibrinogen enriched preparation using any of a range of techniques well known to those skilled in this area. For example, purifying the fibrinogen from the fibrinogen enriched preparation by either reprecipitating the fibrinogen with a protein precipitant in the presence of salts and/or amino acids or by chromatographic techniques such as ion exchange, affinity, hydrophobic or gel permeation chromatography or a combination of both techniques. For use the fibrinogen enriched preparation will typically be treated to remove SPS and/or plasminogen. This can be achieved using a number of methods well known in the art. Examples of known purification methods include those described in the following references, the disclosures of which are incorporated herein by reference:
xe2x80x9cAffinity purification of human fibronectin on immobilized gelatinexe2x80x9d Regnault V, Rivat C, and Stoltz; Journal of Chromatography, 432 (1988) 93-102
xe2x80x9cIsolation of Fibronectin under Mild Conditionsxe2x80x9d Morgenthaler J, Baillod P and Friedli H; Vox Sang 47 (1984) 41-46
xe2x80x9cPlasminogen: Purification from human plasma by affinity chromatographyxe2x80x9d Deutsch D and Mertz E; Science 170 (1970) 1095-1096
xe2x80x9cA Pasteurised Concentrate of Human Plasma Factor XIII for Therapeutic Usexe2x80x9d Winkelman L, Sims G, Haddon M, Evans D and Smith J; Thrombosis and Haemostasis 55(3)(1986) 402-405
xe2x80x9cThe Preparation of Human Fibrinogen Free of Plasminogenxe2x80x9d Mosesson M; Biochim Biophys Acta 57 (1962) 204-213
U.S. Pat. No. 3,340,156
xe2x80x9cSeverely Heated Therapeutic Factor VIII Concentrate of High Specific Activityxe2x80x9d Winkelman L, Owen n, Evans D, Evans H, Haddon M, Smith J, Prince P and Williams J; Vox Sang 57 (1989) 97-103
xe2x80x9cPlasma Protein Fractionationxe2x80x9d Heide K, Haupt H and Schwick H; in The Plasma Proteins, 2nd Edition Vol 3 (1977) Putnam F. (Ed)
Depending on the nature of the fibrinogen containing solution the fibrinogen enriched preparation may also contain fibronectin and Factor XIII. For example, if the fibrinogen containing solution is plasma or a plasma fraction the fibrinogen enriched preparation will also contain fibronectin and/or Factor XIII. If desired these proteins may also be further purified from the fibrinogen enriched preparation using known separation techniques.
Accordingly, the present invention also provides a method of obtaining a preparation enriched for fibronectin or Factor VIII, the method comprising extracting fibronectin or Factor VIII from the fibronectin enriched preparation obtained according to the method of the present invention in which the fibrinogen containing solution is a blood plasma fraction.
As will be recognised from the above description the present invention provides a method of purifying fibrinogen from blood plasma concentrates especially cryoprecipitate. The most commercially important of the plasma concentrates currently used are the blood plasma fraction commonly known as cryoprecipitate and purified concentrates prepared from cryoprecipitate. Conventionally, cryoprecipitate is defined as a precipitate rich in Factor VIII and fibrinogen and which is prepared from frozen freshly prepared human plasma by a low temperature plasma fractionation technique.
Typically deep frozen plasma is softened at temperatures below 5xc2x0 C. and the Factor VIII rich cryoprecipitate is collected by centrifugation.
Cryoprecipitate prepared in this way has been used as a commercial source of Factor VIII and typically contained concentrated within it from 40 to 60% of the total amount of Factor VIII contained in the whole blood from which the plasma is derived. There have been numerous studies designed to improve the yield of Factor VIII from cryoprecipitate and to further purify it. The presence of high concentrations of fibrinogen and fibronectin in Factor VIII preparations is undesirable because they have been found to give rise to unacceptable losses of Factor VIII during some of the processing steps. Fibrinogen is of particular concern because it is normally present in much greater concentrations than fibronectin in blood plasma and cryoprecipitate and is usually more difficult to remove than fibronectin.
A method of preparing a Factor VIII containing preparation which includes the steps of precipitating fibrinogen and fibronectin from a buffered solution of a Factor VIII containing blood plasma fraction by the addition of a sulphated polysaccharide is disclosed by Winkleman (AU B55435/86) who described one method of purifying Factor VIII in which the fibrinogen is precipitated from a buffered solution of cryoprecipitate held at pH 6 to 8.
The heparin precipitate is removed from the Factor VIII containing supernatant and discarded. The present invention enables the purification of valuable proteins from this previous waste product.